Composite

Part:BBa_K2507008

Designed by: Huanding Ji   Group: iGEM17_SHSBNU_China   (2017-10-07)
Revision as of 11:58, 31 October 2017 by HaoyuZhou (Talk | contribs)


J23105-thsR-PphsA342-sfGFP

Usage and Biology

E.coli codon optimized ThsS(BBa_K2507000) and ThsR(BBa_K2507001) are two basic parts which belong to the two-component system from marine Shewanella halifaxensis. ThsS is the membrane-bound sensor kinase (SK) which can sense thiosulfate outside the cell and ThsR is the DNA-binding response regulator(RR). PphsA(BBa_K2507018) is a ThsR activated promoter when ThsR is phosphorylated by ThsS after ThsS sensing thiosulfate. Because of thiosulfate is an indicator of gut inflammation (Levitt et al, 1999; Jackson et al, 2012; Vitvitsky et al, 2015), this system can be used as a sensor of gut inflammation.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 11
    Illegal NheI site found at 34
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1313

Characterization

After validated this system in laboratory Escherichia coli Top10 and E.coli Nissle 1917, this system worked as a thiosulfate sensor. Link thsR with sfgfp (BBa_K2507008), chromoprotein genes (BBa_K2507009, BBa_K2507010, BBa_K2507011) or vioABDE(BBa_K2507012), this system can response to thiosulfate by produce sfGFP, chromoproteins (spisPink-pink chromoprotein, gfasPurple-purple chromoprotein, amilCP-blue chromoprotein) or dark-green small molecular(protoviolaceinic acid).

File:SHSBNU 17 40a01.jpg
alt text

Figure 1. Schematic of ligand-induced signaling through ThsS/R and plasmid design of the sensor components. ThsS/R were tested under the situation BBa_K2507004 was in pSB4K5 backbone and BBa_K2507008 was in pSB1C3 backbone. We submitted the parts all to the iGEM registry in pSB1C3.

We first tested whether the system work. Characterization experiments were performed aerobically. Bacteria were cultured overnight in a 96-deep well plate, 1ml LB media +antibiotics+different concentration of inducer(thiosulfate).

The conclusion is the system(ThsS/ThsR) works, while the leakage is very heavy.

alt text

Figure 2. Characterize thsS/R system by sfGFP expression level. We add 1mM,0.1mM,0.01mM and NA Na2S2O3, it shows response while the leakage is heavey.

Previously, Schmidl et al have shown that thsR overexpression in the absence of the cognate SK and input can strongly activate the output promoter (Schmidl et al, 2014), possibly due to RR phosphorylation by alternative sources (small molecules, non-cognate SKs), or low-affinity binding by non-phosphorylated RRs. We thought that our thsR overexpression is originate from pSB4K5 which have several mutation at pSC101 sequence. It means pSB4K5 is actually a high-copy plasmid! 

https://parts.igem.org/Part:pSB4K5:Experience

Due to the limited time, we didn’t have time to change the backbone to another low copy number plasmid, while we would try after iGEM Jamboree 2017. Then, We characterize the system at aerobic and anaerobic condition. We measured sfGFP intensity by flow cytometry.(Protocol的链接).The response curve in aerobic and anaerobic condition seems

alt text

Figure 3. We characterized ThsS/R system in E.coli Top10 and E.coli Nissle 1917 by sfGFP expression level measured by flow cytometry.

File:SHSBNU 17 40a04.jpg
alt text

Figure 4. We characterized ThsS/R system by flow cytometry.

Reference

Daeffler, K. N., Galley, J. D., Sheth, R. U., Ortiz‐Velez, L. C., Bibb, C. O., & Shroyer, N. F., et al. (2017). Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Molecular Systems Biology, 13(4), 923.

Jackson MR, Melideo SL, Jorns MS (2012) Human sulfide: quinone oxidoreductase catalyzes the first step in hydrogen sulfide metabolism and produces a sulfane sulfur metabolite. Biochemistry 51: 6804 – 6815

Levitt MD, Furne J, Springfield J, Suarez F, DeMaster E (1999) Detoxification of hydrogen sulfide and methanethiol in the cecal mucosa. J Clin Invest 104: 1107 – 1114

Schmidl SR, Sheth RU, Wu A, Tabor JJ (2014) Refactoring and optimization of light-switchable Escherichia coli two-component systems. ACS Synth Biol 3: 820 – 831

Vitvitsky V, Yadav PK, Kurthen A, Banerjee R (2015) Sulfide oxidation by a noncanonical pathway in red blood cells generates thiosulfate and polysulfides. J Biol Chem 290: 8310 – 8320


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